PROJECT SUMMARY/ABSTRACT In our previous studies on occurrence of Legionella and other similar bacteria that can infect amoebae in constructed and natural environments we discovered several amoeba-infecting bacteria that do not grow outside of a host cell; and two, in particular, can infect the nucleus of amoebae. They were determined to constitute a new candidate genus. The two new intranuclear bacteria, ?Candidatus Berkiella cookevillensis? and ?Candidatus Berkiella aquae? were isolated from amoebae of a cooling tower and a hot tub, respectively. ?Ca B. cookevillensis? is capable of infecting the nuclei of mammalian cells, whereas ?Ca. B. aquae? is limited to amoebae. The pathways the novel bacteria utilize to traffic through host cells and into the nuclei, where they replicate, are unknown. The serendipitous recovery of these two, closely-related bacteria has provided us with a unique model system to study the intracellular pathways of these bacteria. Our goal is to understand the details of the pathways of these bacteria into nuclei. Progress toward our goal has been hindered by the obligate intracellular nature of these bacteria; however, we now have toolsets to further our efforts as we have 1) sequenced and annotated the genomes of both bacteria, and 2) have produced polyclonal antibodies to each bacterium. One specific AIM is to determine the intracellular movement of ?Ca. B. cookevillensis? and ?Ca. B. aquae? through host cells and into the nucleus. This AIM will be addressed by using transmission electron microscopy and confocal microscopy of stained live and fixed infected hosts. Immunostaining of endosomal markers and other cellular membrane proteins, along with cytological stains such as FM4-64, LysoTracker, and ER-Tracker will be used. Another AIM is to analyze sequenced genomes for effectors involved in intracellular trafficking and growth. Web-based prediction tools will be used to predict the presence of Dot/Icm secreted effector proteins. In addition, bacterial transcriptome analyses will be done using RNA sequencing. The Illumina Hiseq platform will be used to carry out sequencing, and the R package edgeR will be used to perform differential expression analyses. The ultimate outcome, beyond the reach of the present study, would be to advance medicine by utilizing mechanisms identified in our novel species to develop protocols for targeted transport of molecules into cell nuclei, such as for treatments to interfere with replication of virally-infected or cancer cells. This work addresses NIH?s mission of seeking fundamental knowledge of the nature and behavior of living systems and applying that knowledge to enhance health and reduce illness.